Efficient reamplification of differential display products by transient ligation and thermal asymmetric PCR.
A new method for specific reamplification of DDRT-PCR products is presented. After transient ligation of the primary DDRT-PCR fragments into a T-vector, the cDNAs of interest were reamplified by hemi-nested PCR and thermally asymmetric cycles. In contrast to the originally described protocol, this method of reamplification is specific, sensitive, reproducibly gives a high yield of DNA and allows direct sequencing of the reamplified product without purification or cloning.
Bonnet
S
S
Unité d'Ecologie des Systèmes Vectoriels, Institut Pasteur, 25 rue du Docteur Roux, 75015 Paris, France.
Prévot
G
G
Bourgouin
C
C
D000818:Animals; D000852:Anopheles / Q000235:genetics / Q000469:parasitology; D001483:Base Sequence; D011088:DNA Ligases; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005069:Evaluation Studies as Topic; D005822:Genetic Vectors; D021141:Nucleic Acid Amplification Techniques; D010963:Plasmodium falciparum / Q000254:growth & development / Q000472:pathogenicity; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D011088:DNA Ligases